Quantifying changes in umbilicus size to estimate the relative age of neonatal blacktip reef sharks (Carcharhinus melanopterus)

Live-bearing shark species are inherently born with an open wound, the umbilicus. The healing status of this umbilicus can be used to estimate the age of newborn sharks. This study proposes the first umbilical wound classification based on quantitative changes in umbilicus size allowing accurate age estimates of newborn sharks.


Introduction
The world's oceans are rapidly changing (Halpern et al., 2008;Hoegh-Guldberg and Bruno, 2010;Cooley et al., 2022). Exposure to environmental and anthropogenic stressors, such as those associated with human-driven climate change, fisheries or pollution, may profoundly affect development and growth of elasmobranch fishes (i.e. sharks and their relatives) that are critical to the health of marine ecosystems (Rosa et al., 2014;Wheeler et al., 2021). Growth rates make up an important part of a species life history but also represent trade-offs with other life history traits (e.g. survival and reproduction). Although a diverse range of growth patterns can be found in sharks and their relatives, they typically exhibit K-selected life history strategies, characterized by slow growth rates, late maturation, low fecundity and high longevity (Pianka, 1970;Cortés, 2000). Their slow growth and associated inherent vulnerability to fishing emphasize the importance of understanding these life history characteristics. Indeed, accurate length-at-age descriptions in growth models provide valuable information for assessing population sizes and demographic processes and are crucial to understand productivity, fisheries stock status, maximum sustainable yields and population extinction risks. However, life history data of neonatal sharks, particularly sizes at birth, are often missing from such models, leading to biased growth parameters and rendering their utility less ecologically relevant (Haddon, 2011;Pardo et al., 2013). The collection of morphometric data in neonatal sharks, combined with accurate aging of the neonates, is therefore essential to assess the impacts that chronic stressors may have on future generations, given that early-life stages are some of the most vulnerable.
Fixed length-at-birth (L 0 ) values based on estimated, as opposed to observed, measurements have frequently been incorporated in growth curves to correct for missing dataoften as a result of gear selectivity-on smaller individuals (Pardo et al., 2013). This approach, however, underestimates measures of uncertainty (e.g. standard deviations) and can substantially increase growth estimate bias (Pardo et al., 2013). Indeed, sizes at birth are generally highly variable in neonatal sharks, often depending on the amount of energy invested by the mother during embryonic development (Hussey et al., 2010;Weideli et al., 2019a). Moreover, growth rates in the first few years of life are significantly faster than growth rates of adults, with further differences between males and females; yet, adults exhibit some of the slowest growth rates of all vertebrates (Werner and Griebeler, 2014). Such asymmetries discredit a fixed L 0 and preclude accurate length-at-age estimates, thus emphasizing the need to include empirically observed data from neonates to compliment juvenile and (sub)adult growth rates for models that are key to conservation and management (Smart et al., 2015).
Despite substantial knowledge gaps regarding early-life stage morphometric data, it is evident that neonatal sharks have to effectively manage their energy resources to optimize growth while maximizing survival. Structural damage to tissues originating from various sources, including predation and human activities Hussey et al., 2017), may however lead to energy being diverted away from routine metabolic activities (e.g. growth and foraging) toward healing processes to restore homeostasis. Wound healing is therefore an important process, especially for early-life stages where open wounds are potential sources of infection that may cause complications in neonatal sharks that are still developing their immune system . Elasmobranchs, in particular, exhibit remarkable wound healing throughout their lives, such as the high healing capabilities observed in juvenile blacktip reef sharks (Carcharhinus melanopterus; Chin et al., 2015) and adult whale sharks (Rhincodon typus; Womersley et al., 2021), and it is thought that they consistently show a high capacity for wound healing throughout their lives Womersley et al., 2021). In viviparous species (i.e. those that bear live young), which make up 58% of all elasmobranchs (Compagno, 1990;Dulvy and Reynolds, 1997), the most prominent non-inflicted wounds are at the umbilicus of neonates. Umbilical wounds remain open (i.e. underlying muscle tissue remains visible) until ∼1-2 months post-parturition, depending on the species' life history (Castro, 1993;Ulrich et al., 2007;Chin et al., 2015). As such, rapid wound healing at a predictable and consistent site could be used for estimating neonatal life stage and relative age in sharks (Duncan and Holland, 2006;Chin et al., 2015).
The umbilicus has been used extensively to classify neonates of the viviparous Requiem sharks (family Carcharhinidae) into categories based on the umbilical wound healing status (e.g. Duncan and Holland, 2006;Aubrey et al., 2007;Hussey et al., 2010;Marie et al., 2017;Weideli et al., 2019a). However, to date, such studies have made use of classifications based on subjective categories (e.g. 'open', 'partly healed' and 'recently closed') rather than quantitative changes derived from recaptured individuals. Objective umbilicus size classifications for viviparous shark neonates are needed to interpret and compare early-life characteristics between studies, species, across populations and with respect to environmental and anthropogenic factors. Using more accurate length-at-age data, based on quantitative changes in umbilicus size, instead of pooling length measurements in yearly bins (e.g. all neonates and young-of-the-year juveniles in one age-0 group), may in addition considerably increase the model fit of age and growth curves, and hence, their biological and ecological relevance. This may be particularly important for neonatal and juvenile sharks considering their generally high growth rates (Parsons, 1985). Indeed, growth rates decrease monotonically with age when considering a von Bertalanffy growth curve, the most widely applied growth function (Harry et al., 2022).
The ability to quantitively estimate the age of neonatal sharks may also allow for a more detailed assessment of neonatal mortality rates. Peterson and Wroblewski (1984) . developed an age-specific equation to estimate natural mortality rates from weight-at-age data that could be applied to the different umbilical wound classes (UWCs). Indeed, this method has previously been used to estimate the transition in natural mortality rates from young-of-the-year to adult blacktip (C. limbatus; Heupel and Simpfendorfer, 2002) and bull (C. leucas; Heupel and Simpfendorfer, 2011) sharks; although this indirect technique may underestimate actual mortality rates. Direct estimates through telemetry likely produce more biologically reasonable representations of early-life mortality rates Simpfendorfer, 2002, 2011). A finer resolution of age-specific mortality will further elaborate on the survival dynamics in neonatal sharks. Weight loss during the first few months-post-parturition-has, for instance, been cited as an important cause of neonatal mortality (Duncan and Holland, 2006;Hussey et al., 2010;Corsso et al., 2018;Weideli et al., 2019a). However, the use of subjective categories of umbilical wound healing status and the inconsistency among number of applied classes in previous studies, generally ranging from three to five classes (e.g. Duncan and Holland, 2006;Aubrey et al., 2007;Hussey et al., 2010;Marie et al., 2017;Weideli et al., 2019a), make it difficult to compare these data among species and across populations. Furthermore, earlier published qualitative umbilical wound classifications often lack actual 'time zero' sharks (i.e. neonates bearing remnants of the umbilical cord implying recent birth, hereafter referred to as 'days-old neonates'; e.g. Figure 1), likely due to the small probability of catching these animals within a time span of a few days.
Being able to accurately age neonatal sharks can be a valuable tool for estimating the period and peak of parturition seasons, which represents fundamental knowledge for effective, species-specific fisheries management. However, data on days-old neonates are scarce, and direct observations of parturition in the wild are rare. The period of parturition has therefore often been estimated based on reappearances of newly slender female sharks, for which recent observations indicated pregnancy (Porcher, 2005;Mourier et al., 2013a). However, this method requires intensive monitoring of the adult population via many hours of underwater surveying, and female reappearance may take several weeks. Alternatively, length measurements in combination with embryonic development times and formation of the birth zone in vertebral centra have been used to back calculate the time of parturition (Hall et al., 2012;Santander-Neto et al., 2020). Length measurements are, however, not a suitable indicator of age due to the systematic size overlap across age classes (Weideli et al., 2019a;this study), and studying vertebral centra is only possible in deceased animals. A more convenient and non-lethal method would be to use regression relationships between umbilicus healing rate and the age of neonatal sharks to trace back the timing of parturition.
The primary objective of this study was to quantify changes in umbilicus size of neonatal blacktip reef sharks (C. melanopterus) around the island of Moorea, French Polynesia, based on the temporal regression relationships of umbilicus area and perimeter. The abundance of neonatal and juvenile reef sharks on the shallow reefs fringing the island, which serve as parturition areas, make it an ideal location to collect early-life history data and study the population dynamics of these sharks (Mourier and Planes, 2013;Mourier et al., 2013b;Chin et al., 2015;Bouyoucos et al., 2020Bouyoucos et al., , 2022. Furthermore, previous research on the wound healing capabilities of blacktip reef sharks make this a well-suited species to address the issues set forth here , and their extensive distribution throughout the Indo-Pacific (Ebert et al., 2021) may offer the potential for widespread applicability of the proposed classification. The secondary objective was to test and validate the accuracy of our umbilical wound classification to elaborate on the utility of quantitative classifications as the one proposed here, and we provide two examples to illustrate their efficacy. Considering the rapid growth and development of neonatal sharks (Randall, 1977;Gruber and Stout, 1983;Branstetter, 1990;Freitas et al., 2006;Weideli et al., 2019b), neonatal age estimates are crucial data for accurate lengthat-age descriptions that are fundamental in understanding productivity, fisheries stock status and risk of population loss and can help further our understanding of early-life growth rates, periods of parturition, initiation of successful feeding, neonatal survival dynamics and the use of parturition areas. As such, this tool provides an absolute needed alternative to lethal sampling for the purpose of age estimates in neonatal sharks.

Methods and Materials
All shark capture and research protocols were approved under Arrêté N • 9524 issued by the Ministère de la Promotion des Langues, de la Culture, de la Communication et de Neonatal and juvenile blacktip reef sharks were caught using a 50 × 1.5 m gillnet with 5-cm mesh size set perpendicular to shore. Gillnets were set at dusk from ∼1700 to 2000 h at ten sites (Apaura, Haapiti, Maharepa, Paorea, Papetoai, Pihaena, Tiki, Vaiane, Vaiare and Valorie). Sites were evenly spread out around the 60-km coastline of Moorea, with each site sampled twice per month (e.g. Mourier and Planes, 2013;Mourier et al., 2013b;Chin et al., 2015;Bouyoucos et al., 2020Bouyoucos et al., , 2022. On capture, sharks were tagged with internal passive integrated transponder (PIT) tags and/or external T-bar anchor tags to allow for the identification of previously caught sharks, their umbilicus was photographed and morphometrics were taken. A ruler was photographed beside each umbilicus for scale (see Figure 1). For the purpose of this study, sharks that have an open umbilicus (i.e. with visible muscle tissue, see Figure 2c) are referred to as neonates, whereas those with a fully healed umbilicus (i.e. completely closed skin) are referred to as (young-of-the-year) juveniles.

Quantifying changes in umbilicus size
Sixteen individual days-old neonates ('time zero' animals) caught between September 2016 and February 2022-of a total of 727 caught individuals-that were subsequently recaptured during the following weeks were considered for quantifying changes in umbilicus size over time. Neonatal blacktip reef sharks lose the remnants of their umbilical cord within the first couple of days post-parturition (S. Debaere, personal observation), and these remnants are therefore a good indicator of recent birth. To assess the size of the umbilicus of neonatal sharks, photographs were imported in the open-source image processing package Fiji by ImageJ (version 2.0.0-rc-69/1.53c; Schindelin et al., 2012). Scale was set to 1 cm using the ruler in the photographs, and the circumference of the umbilical wound was carefully traced using the polygon selection tool from ImageJ to calculate umbilicus area and perimeter. Temporal closing of the umbilicus of the daysold neonates and their subsequent recapture (13 individuals were recaptured once, 3 individuals were recaptured twice) allowed for a quantitative classification of the umbilicus of the sharks into four categories, or UWCs, based on the area and perimeter of the umbilicus at four time intervals. These four UWCs include three distinct neonatal UWCs (i.e. open/unhealed umbilicus, in order of increasing age: UWC1, UWC2 and UWC3) and one juvenile class (i.e. closed/healed umbilicus: UWC4).  (x)) 2 ) with a rapid decrease in area during the first 12 days of age. (b) UW perimeter of recaptured neonates follows a negative linear trend (y = 1.299-0.037 x). The dashed horizontal lines at (a) 0.016 cm 2 and (b) 0.41 cm represent the critical values at 12 and 24 days of age, respectively, used for the classification of the umbilicus size into four categories, or UWCs (see Table 1). Individuals Cm_D, Cm_J and Cm_M were recaptured twice; the other sharks were only recaptured once. (c) Representative photographs of the umbilicus for each UWC with their corresponding UW area (A) and perimeter (P). Scale bar = 1 cm.

Morphometrics and body condition
Morphometric data were collected to assess transitions in growth and body condition across UWCs to shed light on early-life growth patterns and energy reserve depletion rates. Morphometric measurements taken during collections (see preceding section) included total body mass (M, in kilograms) and precaudal length (PCL, distance from the tip of the snout to the origin of the caudal fin, in centimeters). In addition, Fulton's body condition factor (K, sensu Ricker, 1975 (1) Before statistical analyses, where applicable, data were checked for normality using the Shapiro-Wilk test, where the test statistic W < 0.90 was considered as the critical value to reject the null hypothesis that the data come from a normal distribution. The critical value of statistical significance was set to α = 0.05, and all statistical analyses were carried out in RStudio (version 1.3.1093; RStudio Team, 2020; R Core Team, 2020) using core R packages. We used oneway analyses of variance (ANOVAs) to test for significant differences in body mass, precaudal length or body condition among UWCs. When the initial ANOVA found a statistically significant difference in means, the response variables were compared among the four UWCs using Tukey honest significant difference (HSD) test for multiple comparison.
Note that no discrimination was made between sex because blacktip reef sharks do not sexually mature until they reach a total body length of 105-133.5 cm (between the ages of 4 and 8 years, males and females, respectively) (Stevens, 1984;Chin et al., 2013;Mourier et al., 2013b). Furthermore, previous studies found no significant difference in PCL between male and female neonatal and juvenile blacktip reef sharks (Stevens, 1984;Papastamatiou et al., 2009).

Back calculating time of parturition
The approximate date of parturition was back calculated for 452 neonatal sharks by subtracting the mean estimated age at the given UWC from the initial capture date using the formula: where t 0 is the estimated date of parturition (in Julian days), t i is the initial capture date (in Julian days) and t min(UWCi) and t max(UWCi) are the ages (in days) at the lower and higher bounds of the UWC assigned to the shark on initial capture (as provided in Table 1, column 'Estimated age'). Sharks with a closed umbilicus at initial capture (i.e. juveniles) were not considered in these calculations. The distribution of number of parturitions was compared among months using Pearson chi-square test.

Quantifying changes in umbilicus size
Temporal closing of the umbilicus of recaptured neonates (n = 16) suggests a quadratic decline of umbilicus area (fit using the linear mixed-effects lmer function from the lme4 package, with shark identity as random factor, after square root transformation; Bates et al., 2015; Figure 2a) and a negative linear relationship between umbilicus perimeter and time at liberty (TAL) (fit using the lmer function with shark identity as random factor; Figure 2b). Based on these regression relationships, umbilical wounds close completely by 36 days post-parturition (see x-intercept in Figure 2b). Critical values for umbilicus area and perimeter were subsequently chosen at 12, 24 and 36 days to obtain a four-point classification with similar time intervals (Figure 2, Table 1). Note from Table 1 that both parameters are needed to assign a UWC to an individual. The rapid decline in umbilicus area allows for distinguishing UWC1 animals from other classes, but umbilicus area appears to overlap between UWC2 and UWC3. The use of umbilicus perimeter is therefore required in addition to area measurements to distinguish the latter two classes.
To validate the accuracy of the temporal regression analyses, the obtained equations were transformed to allow age to be estimated, in days, for a subset of recaptured sharks (via umbilicus area: age = [−(sqrt(area)−0.311)/0.053] 2 ; via umbilicus perimeter: age = [perimeter−1.299]/[−0.037]). The estimated age of neonates (i.e. the mean of the ages inferred from the temporal regression relationships of umbilicus area and perimeter) at initial capture and recapture was subsequently used to predict the elapsed time between initial capture and recapture, referred to as the predicted time at liberty (pTAL). The pTAL was then compared with the actual TAL to validate the accuracy of the temporal regression analyses. Individuals with a closed umbilicus at initial capture and/or recapture and those that were used to construct the regression relationships were omitted from this comparison. On average, the pTAL differed from the actual TAL by 5 ± 3 days (mean ± standard deviation; n = 17) ( Table 2).  Table 2: Validation of the accuracy of the constructed UWC. For a subset of recaptured sharks, the pTAL was calculated from the temporal regression relationships of umbilicus area and perimeter. Actual TAL between initial capture and recapture was subsequently compared to this pTAL value (|TAL − pTAL|). The mean difference (± standard deviation) between TAL and pTAL is provided in the final row. neonates and juveniles, respectively. Mass ranged from 0.430 to 1.525 kg (0.995 ± 0.162 kg) and 0.560 to 2.400 kg (1.055 ± 0.251 kg) in neonates and juveniles, respectively. Both PCL (F 3,845 = 28.78, P < 0.001; UWC1-UWC4, P < 0.001; UWC2-UWC4, P < 0.001; UWC3-UWC4, P < 0.001; Figure 3a) and mass (F 3,817 = 5.66, p < 0.001; UWC1-UWC4, P = 0.003; UWC2-UWC4, P = 0.048; Figure 3b) showed a positive relationship with increasing UWC, albeit non-significant among the first three UWCs, whereas body condition (Fulton's K; F 3,816 = 25.57, P < 0.001; UWC1-UWC2, P = 0.003; UWC1-UWC3, P = 0.030; UWC1-UWC4, P < 0.001; UWC3-UWC4, P = 0.009; Figure 3c) significantly decreased with UWC.

Back calculating time of parturition
The approximate date of parturition was back calculated for neonatal sharks (i.e. UWC1-3) by subtracting the mean estimated age at a given UWC from the initial capture date (i.e. UWC1 -6 days; UWC2 -18 days; UWC3 -30 days). We observed a clear association between parturition month and the number of neonatal sharks (χ 2 = 249.13, P < 0.001; Figure 3d, left y-axis, light grey bars). For the blacktip reef shark population around the island of Moorea, the parturition season starts in September (5.2% of total parturitions), but most parturitions occur during October (32.7%) and November (41.3%), after which the number of births decreases during December (17.6%), and only a few neonates were assumed to have been born in January (3.2%). A similar trend but with 1-month delay can be observed in the total number of sharks caught throughout the parturition season ( Figure 3d, right y-axis, dark grey bars).

Discussion
The present study proposes the first umbilical wound classification based on quantitative changes in umbilicus size, a non-lethal alternative to aging neonatal sharks, thus allowing the relative age of neonatal sharks to be estimated. This precise tool will help estimate neonate abundances and inform the use of spatial or temporal fishery closures. Accurate age estimates of neonatal sharks are essential data to further our understanding of their early-life characteristics and environmental and anthropogenic impacts thereupon. As such, this study contributes valuable data to inform the conservation and management of young-of-the-year blacktip reef sharks in French Polynesia, information which could be applied to other populations and species globally. The temporal regression relationships provided in this study for umbilicus area and perimeter are similar to the changes in umbilicus size reported by Chin et al. (2015)  healing of blacktip reef sharks in a laboratory setting. However, Chin et al. did not follow umbilical wound healing in days-old neonates, thus preventing the use of their data for estimating age. Yet, the ability to quantitatively estimate the age of neonatal sharks is fundamental knowledge that can help infer the depletion rate of maternally provided in utero energy reserves, subsequent onset of successful feeding and the timing of parturition, as well as many other milestones that are key to early-life history.
Learning how to forage effectively is one of the primary challenges neonatal sharks encounter after parturition (or hatching in oviparous species). Viviparous sharks receive maternal energy reserves in the form of enlarged livers that sustain neonates early in life (Hussey et al., 2010). Once these reserves are depleted, the sharks must start feeding to compensate for the energetic costs of life. Our data show a marked decrease in body condition after age of 12 days (UWC2), suggesting depletion and insufficient replenishment of these energy reserves. Indeed, previous studies have already demonstrated low food consumption rates in coastal youngof-the-year sharks and a reduction in body condition during the first weeks to months post-parturition (Bush and Holland, 2002;Lowe, 2002;Duncan and Holland, 2006;Weideli et al., 2019a). Weideli et al. (2019a) reported that the rate of successful feeding (based on the proportion of empty stomachs to stomachs containing prey items) increases with umbilical wound stage (UWC1, 30% of the stomachs contained prey items; UWC2, 47% of the stomachs contained prey items and UWC3, 51% of the stomachs contained prey items), but their data also clearly demonstrate that the sharks seem to feed at insufficient rates (e.g. due to limited prey availability), making it difficult in this case to inform on the onset of successful feeding. However, in areas where prey items are abundant and easily accessible, the onset of successful feeding can be more readily quantified. Similar to our results, Weideli et al. (2019a) found a significant decrease in body condition with increasing UWC in the same study population around the island of Moorea but only between young-of-the-year juveniles (i.e. sharks with fully healed umbilicus) and first-class neonates (i.e. sharks with fully open umbilicus). The quantitative four-point classification applied in our analyses (i.e. UWC1-4), as opposed to the qualitative three-point classification used by Weideli et al. (2019a) (where UWC1 corresponds to individuals with a 'fully open umbilicus'; UWC2, 'semi-healed umbilicus' and UWC3, 'fully healed umbilicus'), allowed for more precision in transitions in body condition and suggests a decrease in condition occurs as early as 12 days post-parturition. The in utero-allocated energy reserves, therefore, likely only sustain the neonates during the first 2 weeks, rather than month ( these depletion rates are likely species-and context-specific). A further decrease in body condition observed in the juvenile age class (UWC4) may be the result of limited prey availability or quality, variable foraging strategies and the negative allometric growth reported for the species (i.e. faster increase in length than in mass; Weideli et al., 2019a).
We observed the largest variations in PCL, mass and body condition in the juvenile age class (UWC4). The UWC4 comprises all young-of-the-year sharks with a healed umbilical wound, and the time interval in this class is therefore much larger (i.e. a resolution of months) than that of UWC1-UWC3 (i.e. 12 days each). Animals born during a previous parturition season (i.e. +1-year-old sharks) were rarely recaptured (i.e. only 6 sharks were recaptured in a subsequent season, based on tag presence) and were therefore excluded from the young-of-year age class. The large variation in morphometric data in UWC4 are therefore likely a result of the variable growth rates in this class (Weideli et al., 2019a(Weideli et al., , 2019b. In study populations where recapture rates are high, estimates of neonatal age may allow juvenile age of recaptured individuals to be inferred from the elapsed TAL since initial capture, and thus allow for distinct classifications of juvenile groups. However, in our study population around Moorea, we had a recapture rate of ∼8.5%, which was too low to get sufficiently large sample sizes if we were to split the juvenile group into more defined classes with similar time intervals to UWC1-3 (i.e. ∼12 days). The low chance of recapturing individuals on the reefs that fringe Moorea may be due to high neonatal mortality rates together with an expansion in foraging area as the sharks grow. High recapture rates that would allow for distinct juvenile age classes may be more easily obtained from naturally enclosed parturition areas (e.g. Gruber et al., 1988Gruber et al., , 2001 and something to consider for future investigations and for other species. In addition, in areas where recapture rates are inherently low, neonatal sharks with an actively healing umbilicus could be maintained in sea pens to track healing rates to allow for the construction of similar temporal regression relationships of umbilicus size. Determining the timing of parturition is critical information for the effective management of shark populations and can be done if neonatal shark ages can be estimated. Back calculating the time of birth in this study suggests that the parturition season of the blacktip reef sharks around the island of Moorea starts in September and lasts until January, with most parturitions occurring during October and November. Our data for Moorea's blacktip reef shark population provide similar results to those found by Porcher (2005) regarding the progression of adult females through pregnancy, further corroborating the efficacy of our classification.
The accuracy of the proposed umbilical wound classification is further supported by the predictions of elapsed time between initial capture and recapture (pTAL) inferred from the age of the sharks. The error rate of pTAL relative to the actual elapsed TAL of 5 ± 3 days is well within range of our proposed UWCs with a resolution of 12 days. Nevertheless, considering the rapid growth and development of neonatal sharks (Randall, 1977;Gruber and Stout, 1983;Branstetter, 1990;Freitas et al., 2006;Weideli et al., 2019b), an error rate of ∼1 week may be considerable. Indeed, a lot may happen during the first weeks of a shark's life, from learning how to forage (Guttridge et al., 2009;Weideli et al., 2019a), evade predation (Guttridge et al., 2012;Hussey et al., 2017;Trujillo et al., 2022), compete with other neonates (Matich et al., 2017) and cope with anthropogenic stressors (Knip et al., 2010)-stressors that may, in turn, affect healing rates-to the active development of their immune system (Rumfelt, 2014). We therefore encourage the use of UWCs, rather than daysof-age values, to minimize the impact of these error rates.
It is also important to note that umbilical wound classification is likely species- (Castro, 1993;Ulrich et al., 2007) and region-specific because physiological processes of ectotherms, such as wound healing, directly depend on ambient temperature regimes (e.g. Anderson and Roberts, 1975;Smith et al., 1988;Pressinotti et al., 2013;Jensen et al., 2015). Higher environmental temperatures, up to the point of thermal stress, may accelerate umbilicus healing rates (Chin et al., 2015, Debaere et al., unpublished data) and thereby influence the regression lines. We therefore encourage comparisons of umbilical wound healing rates across populations to inform the potential for widespread applicability of the proposed classification. To elaborate on the differences in umbilicus healing rates among species, we strongly recommend similar regression relationships as those provided here to be constructed for other viviparous shark species.
In summary, this study is the first to propose an age classification for shark neonates based on quantitative changes in umbilicus size. Our temporal regression relationships of umbilicus area and perimeter allow the relative age of neonatal blacktip reef sharks to be estimated and grouped into distinct UWCs. The accuracy of our umbilical wound classification is supported by the minimal error rates observed between predicted and actual TALs of recaptured neonates. Nevertheless, considering the rapid development of neonatal sharks, we encourage the use of distinct UWCs, rather than their actual age, to minimize the impact of these error rates. Neonatal age estimates are essential data for accurate lengthat-age descriptions that are fundamental for understanding productivity, fisheries stock status and population extinction risks. Therefore, these data can help further our understanding of early-life growth rates, neonatal survival, use of parturition areas, essential habitats and ontogenetic shifts in home ranges. Indeed, our data illustrate the efficacy of quantitative classifications of umbilical wound healing status for inferring periods of parturition and highlight how rapidly maternal energy reserves that were provided in utero deplete and the delayed onset of successful feeding. Overall, this study contributes valuable data to inform the conservation and management of young-of-the-year blacktip reef sharks in French Polynesia and provides a detailed description for the construction of similar quantitative UWCs for other species.

Data Availability Statement
The data underlying this article are available in the Zenodo repository, at https://dx.doi.org/ 10.5281/zenodo.7232179.

Author Contributions
S.F.D. conceived the study; S.F.D., O.C.W., I.A.B., K.B.E., J.E.T., G.D.B. and J.L.R. collected field data; S.F.D. analysed the data and drafted the manuscript. All authors secured funding to support this study, contributed to the editing of the final manuscript, and gave final approval for publication.